Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi

Abstract

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P<0.05) greater mRNA of IFN-gamma, IL-8, IL-12p35, and significantly (P<0.05) decreased TNF-alpha mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P<0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-alpha. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P<0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P<0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-alpha. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.